Figure 11.

GRP78 overexpression or SREBP2 knockdown reduces α-syn aggregation in primary neuronal cultures elicited by nutrient deprivation. Primary cultures with human α-syn overexpression were infected with lentivirus carrying GRP78 or shRNA of mouse SREBP2. 7 days after the infection, cells were maintained in media with or without nutrient deprivation for 24 h. Duplicated cultures in 48 well plates were included for cell viability assessment and cholesterol assay. (A) Cell lysates were probed with antibodies to α-syn, GRP78, CHOP, cleaved Caspase 3, SREBP2 and β-actin. Molecular weight standards were included as references. (B–H) Bar graphs summarized quantitative immunoblot analysis of various proteins from three independent experiments and normalized with β-actin. The average values of Con groups were set as 100% for all other proteins. (I) Calcein assay showing overexpression of GRP78 or knockdown of SREBP2 significantly rescued cell loss elicited by nutrient deprivation. (J) Amplex Red-based cholesterol assay showing GRP78 overexpression or SREBP2 knockdown can prevent nutrient deprivation-induced cholesterol increase in primary cultures. Error bars represent standard error of the mean (* p < 0.05, ** p < 0.01, comparing to Con; # p < 0.05, ## p < 0.01, comparing subsets linked by line). (K) Immunocytochemistry demonstrated the alteration of SREBP2 level in primary neurons (showing neuronal staining with MAP2) of aforementioned six groups. Scale bar: 10 µm. (L) Bar graphs summarized quantitative analysis of nuclear SREBP2 fluorescence intensity from at least 50 immunocytochemically stained primary neurons per group (* p < 0.05, ** p < 0.01, comparing to Con; # p < 0.05, ^## p < 0.01, comparing subsets linked by line).